Glial fibrillary acidic protein (gfap)

Immunofluorescence was performed as previously described in our laboratory25 (link)26 (link). In brief, the tissue sections were deparaffinized, rehydrated and underwent antigen recovery. Then, the sections were permeabilized with 0.4% Triton X-100 for 10 min and blocked using normal goat serum (Zhongshan Golden Bridge Inc., Beijing, China) for 1 h to eliminate nonspecific staining and incubated in a mixture of rabbit anti-NR4A1 antibody (Proteintech), mouse anti-microtubule-associated protein 2 (MAP2) antibody (Zhongshan Golden Bridge) and chicken anti-astrocyte marker glial fibrillary acidic protein (GFAP, Zhongshan Golden Bridge) antibody or goat anti-Aldehyde Dehydrogenase 1 Family Member L1 (Aldh1L1) antibody (Santa Cruz) overnight at 4 °C. Cells were washed using PBS and incubated with Alexa Fluor-350 goat anti-mouse IgG, Alexa Fluor-488 goat anti-rabbit IgG, and Alexa Fluor-594 goat anti-chicken IgG or Alexa Fluor-594 donkey anti-goat IgG (Zhongshan Golden Bridge) in the dark for 2 h at 37 °C. Cells were washed again in PBS, mounted, sealed, and dried overnight. Finally, the images were captured using confocal laser scanning microscopy (Leica, Wetzlar, Germany).

Tissue sections (10 μm) were incubated in normal goat serum (Zhongshan Golden Bridge, Beijing, China) for 30 min followed by incubation with a mixture containing a PDE10A antibody (rabbit polyclonal antibody, 1:100), a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:100, Zhongshan Golden Bridge) and a glial fibrillary acidic protein (GFAP) antibody (mouse polyclonal antibody, 1:100, Zhongshan Golden Bridge) overnight at 4°C. Sections were washed twice in PBS and incubated with a mixture of DyLight 488-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge), DyLight 549-conjugated goat anti-mouse IgG (1:200, Zhongshan Golden Bridge) and DyLight 405-conjugated goat anti-chicken IgG (1:200, Zhongshan Golden Bridge) in a darkroom for 90 min at 37°C. Tissue sections were mounted in 50% glycerol/PBS. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head. To determine the specificity of antibodies, PDE10A antibody (488 channel), GFAP antibody (549 channel) and microtubule-associated protein 2 (MAP2) antibody (405 channel) were replaced by PBS with the same protocols.